A combined biochemical and genetic approach is planned to isolate and characterize mutations in the gene for hypoxanthine phosphoribosyltransferase (HPRT, E.C.2.4.2.8) in human cells. I would particularly like to identify a chain-termination (amber- or ochre-type mutation) or frameshift mutation in HPRT, and then a revertant caused by a suppressor tRNA. These cells, containing suppressor tRNAs, would be of great value for the genetic and biochemical analysis of oncogenic viruses. We are analyzing HeLa and human lymphoblast cells with wild-type, mutant, and revertant forms of HPRT by two-dimensional polyacrylamide gel electrophoresis. In this procedure, proteins are separated first in one dimension by isoelectric focusing, and then in a second dimension by sodium dodecyl sulfate gel electrophoresis. Although HPRT represents only 0.02% of the soluble protein, we have identified the spots corresponding to wild-type and mutant forms of the enzyme. We have devised methods using monspecific antibody against HPRT to prepare apparently homogeneous enzyme protein from cell extracts. We are examining tryptic and cyanogen bromide peptides of wild-type and mutant HPRt proteins by high pressure liquid column chromatography and polyacrylamide gel electrophoresis. We plan to compare the amino acid composition and sequence of wild-type and altered mutant peptides, and from the observed substitutions, deduce the alterations in the nucleic acid sequence in the gene.